The present invention relates to an anti-AIDS therapeutic vaccinal complex, which has vaccination effectiveness connected to the presence of non-specific antigens.
It is well known in bacteriology that the antigens at the surface of walls, membranes or capsules (combined or liberated in soluble form in culture medium) are of glycoproteic, polypeptidic or polysaccharidic nature.
Recent vaccines have been widely publicized, associating with RNA (ribosomal origin) of such proteoglycanic or polysaccharidal membranal substances, extracts of pathogenic germs.
These vaccines use specific antigens corresponding to specifically predetermined microbial diseases.
However, the antigenic power is essentially connected to the RNA level (of the ribosomes in particular) of the microbial cells, among others. The ICC (immunologically competent cells) use directly the RNA as active transporters.
To build our new vaccinal complex, instead of using a serotypical bacterial antigen, we have coupled (by preferably covalent bonds) RNA (preferably of ribosomal origin) to a sequence of amino acids of glycoprotidic nature, present in collagen of type III (in humans, collagen represents approximately one-third of the proteins of the organism; type III has been selected for its amino acid sequence and because it is present essentially in the skin and the vascular walls).
Moreover, RNA is already used in the preparation of acellular vaccines (cf. Infect. and Immunity, 1, 574-82, 1970). This RNA is stabilized by associative factors.
In our complex, we use as stabilizer cellular membrane fractions from the same germs as those which have served for the elaboration of ribosomal RNA. These membranal fractions contain all the peptidalglycanic substances and are known moreover as immunal adjuvants.
Contrary to convention, it is not necessary to have the same membranal fractions (glucopolysaccharidal or proteoglycanic) from the same microbial germs as those which serve to supply RNA by extraction of their ribosomes.
Axe2x80x94Useful RNA of ribosomal origin can be extracted from the following strains, this list not being limiting:
Klebsiella pneumoniae 
Streptococcus (pneumonia and pyogens)
Staphylococcus aureus 
Serratia marcescens 
Escherichia coli 
Salmonella typhimurium 
Corynebacterium (granulosum, parvum, acnes)
Mycobacterium (tuberculosis, smegmatis, chelonei)
Hemophilus influenzae 
Pneumocoque type II
Rothia dento cariosus 
Bacterium coli 
Shigella dysentariae 
Enterococcus
Nocardia (asteroides, brasiliensis, rhodocrans, opaca, rubra)
Bacillus of Calmette and Guerin
The mean molecular weights of these RNAs are between 5,104 and 108 Daltons.
Many industrial processes exist for the preparation of RNA; there will be cited as example the process for the extraction of RNA described in Infect. and Immunity, 1, 574-82, 1970: the bacteria are agitated and then subjected to fractional precipitation, the ribosomal proteins are solubilized, the precipitated RNA is treated with Pronase and finally, purified by ion exchange chromatography.
If RNA is obtained by enzymatic means, the final purification can be effected by molecular sieve chromatography. See particularly on this subject:
C. EHRESMAN (1972)xe2x80x94Biochimie, 54, 901
H. KAGAWA (1972)xe2x80x94J. Biochem., (1972), 827
M. SANTER (1973)xe2x80x94J. Bact., 116, 1304
NOMURA (1974)xe2x80x94Ribosomesxe2x80x94Ed. Cold Spring Harbor Laboratory.
Bxe2x80x94The membranal fractions of usable bacterial cells can be extracted from the following strains, the given list not being limitative:
1 xe2x80x94for capsular polysaccharides
Klebsiella pneumoniae 
Streptococcus pneumoniae 
Hemophilus influenzae 
Escherichia coli 
Klebsiella pneumoniae
C. ERBING, L. KENNE, B. LINBERG, J. LONNGREN (1976)xe2x80x94xe2x80x9cStructural studies of the capsular polysaccharide of Klebsiella pneumoniae type I (Carbohydr. Res., 50 (1976) 115-20).
W. NIMMICH (1968)xe2x80x94xe2x80x9cZur Isolierung und qualitativen Bausteianalyse der K. Angigen von. Klebsiellenxe2x80x9d (Med. Mikrobio. und Immunol., 154 117, 131).
C. RICHARD (1973)xe2x80x94xe2x80x9cEtude antigenique et biochimique de 500 souches de Klebsiellaxe2x80x9d (Ann. Biol. Clin., 1973).
Streptococcus pneumoniae
F. KAUFFMANN and E. LUND (1954) (Int. Bull. Bact. Nomencl. 4, 125-28).
FELTON and OTTINGER (J. of Bacteriology, 1942, 43, 94, 105)
M. COLIN, M. D. MAC LEOD and colleaguesxe2x80x94xe2x80x9cPrevention of pneumococcal pneumoniae by immunization with specific capsular polysaccharidesxe2x80x9d (J. Exp. Med., 1945, 82, 445-65).
A. R. DOCHEZ and O. T. AVERYxe2x80x94xe2x80x9cThe elaboration of specific soluble substance by Pneumococcus during growthxe2x80x9d (1971) (J. Exp. Med. 16, 477-93).
WEST PHAL and LUDERITZ (1952) (Z. Naturf. 7B, 148).
C. P. J. GLAUDEMANS and H.P. TREFFERS, xe2x80x9cAn improved preparation of the capsular polysaccharide from Diplococcus pneumoniae (Carbohydr. Res. 1967, 4, 181-84).
Hemophilus Influenzae (Capsular Polysaccharide of Poly-Ribosphorphate Type)
P. ANDERSON and colleagues (1972)xe2x80x94xe2x80x9cImmunization of humans with polyribosephosphate, the capsular antigen of Hemophilus influenzae type Bxe2x80x9d (J. of Clin. Invest., Vol. 51, 1972, 39-44).
P. ANDERSON and colleagues (1977)xe2x80x94xe2x80x9cIsolation of the capsular polysaccharide from supernatant of Hemophilus influenzae type Bxe2x80x9d (Infect. and Immun., 1977, 15(2), 472-77).
Escherichia coli (Capsular Polysaccharides)
LUDERITZ and colleagues (1977)xe2x80x94xe2x80x9cSomatic and capsular antigens of gram-negative bacteriaxe2x80x9d (Compr. Biochem. 26 A, 105-228).
2xe2x80x94for membranal lipopolysaccharides (LPS) Corynebacterium (avidum, bovis, diphteriae, enzymicum, equi, fascians, flaccum, faciens, flavidum, fusiforme, granulosum, helvolum, hypertrophicans, insidiosum, liquefaciens, parvum, paurometabolum, pyogenes, tumescens, xerosis)
and the gram negative:
Klebsiella (pneumoniae and rhinoscleromatis)
Salmonella typhimurium 
Serratia (marcescens, corralina, indica, polymuthica, kiluea)
Neisseria meningitidis 
Escherichia coli 
C. ERBIN and colleagues (1977)xe2x80x94xe2x80x9cStructural studies on the Klebsiella LPSxe2x80x9d (Carbohydr. Res., 56, 377-81).
C. B. CASTOR and colleagues (1971)xe2x80x94xe2x80x9cCharacteristics of a highly purified pyrogenic LPS of Klebsiella pneumoniae (J. of Pharm. Sci., 60, (10), 1578-80).
K. FUKUSHI (1964)xe2x80x94xe2x80x9cExtraction and purification of endotoxin from Enterobacteriaceae: a comparison of selected methods and sourcesxe2x80x9d (J. of Bacteriol. 87, (2), 391-400).
G. A. LIMJUCOxe2x80x94xe2x80x9cStudies on the chemical composition of LPS from Neisseria meningitidis group Bxe2x80x9d (J. of Gen. Microbiol. 1978, 104, 187-91).
G. A. ADAMS (1967)xe2x80x94xe2x80x9cExtraction of LPS from gramnegative bacteria with DMSOxe2x80x9d (Canad. J. Biochem., 45, 422-26).
K. G. JOHNSON (1976)xe2x80x94xe2x80x9cImproved techniques for the preparation of bacterial LPSxe2x80x9d (Canad. J. Microbiol. (22), 29-34).
Y. B. KIM and colleagues (1967)xe2x80x94xe2x80x9cBiologically active endotoxins from Salmonella mutans (J. of Bacteriol., 94, (5), 1320-26).
3xe2x80x94For Membranal Proteins
Escherichia coli 
Serratia marcescens 
Streptococcus pyogenes 
Salmonella typhimurium 
Escherichia coli 
S. F. STIRM and colleagues (1967)xe2x80x94xe2x80x9cEpisome, carried surface antigen K88 of Escherichia coli (J. of Bacteriol., 93, (2), 731-39).
S. J. BETZ and colleagues (1977)xe2x80x94xe2x80x9cChemical and biological properties of a protein rich fraction of bacterial LPSxe2x80x9d (J. of Immunol., 119, (4), 1475-81).
Serratia marcescens 
W. WOBER (1971)xe2x80x94xe2x80x9cStudies on the protein moiety of endotoxin from gram-negative bacteria, characterization of the protein-moieting isolated by acetic acid hydrolysis of endotoxin of Serratia marcescensxe2x80x9d.
Streptococcus pyogenes 
M. K. WITTNER (1977)xe2x80x94xe2x80x9cHomologous and heterologous protection of mice with group-A Streptococcal M protein vaccinexe2x80x9d (Infect. and Immun., 1977, 15, (1), 104-8).
Salmonella thyphimurium 
N. KUUSI and colleagues (1979)xe2x80x94xe2x80x9cImmunization with major outer membrane protein in experimental salmonellosis of micexe2x80x9d (Infect. and Immun., 1979, 25, (3), 857-62).
C. BARBER and colleagues (1972)xe2x80x94xe2x80x9cThe protective role of proteins from Salmonella thyphimurium in infection of mice with their natural pathogenxe2x80x9d (Rev. Immunol., 36, 77-81).
G. DELORD (1979)xe2x80x94xe2x80x9cEtude d""un antigene vaccinant contenu dans le surnageant de culture de Salmonella thyphimurium, souche M-206xe2x80x9d, Medical Thesis at Lyon, No. 428, 1979.
G. W. GOODMAN (1979)xe2x80x94xe2x80x9cCharacterization of the chemical and physical properties of a novel B-lymphocyte activator endotoxin proteinxe2x80x9d (Infect. and Immun., 1979, 24 (3), 685-96).
4xe2x80x94For Teichoic and Lipoteichoic Acids
Streptococcus, staphylococcus, and lactobacillus (the surface of the Gram-positive bacteria is made of teichoic acid, which is a polymer of glycerol, linked by phosphodiester linkages).
The following articles describe the processes of production:
M. M. BURGER (1966)xe2x80x94xe2x80x9cTeichoic acids: antigenic determinants, chain separation and their location in the cell wallxe2x80x9d (Microbioloqv 56, 910-17).
K. W. KNOX (1973)xe2x80x94xe2x80x9cImmunological properties of teichoic acidsxe2x80x9d (Bacteriol. Reviews, 37, 21, 215-57).
G. A. MILLER (1976)xe2x80x94xe2x80x9cEffects of streptococcal lipoteichoic acid on host response in micexe2x80x9d (Infect. and Immun., 1976, 13, (5), 1408-17).
A. J. WICKEN and colleagues (1975)xe2x80x94xe2x80x9cLipoteichoic acids: a new class of bacterial antigensxe2x80x9d (Science, 187, 1161-67).
Different Possible Dosages
RNA
*FISKE and SUBBAROWxe2x80x94xe2x80x9cDosage du phosphore. Chromatographie HPLC sur colonne echangeuse d""ions pour le controle qualitatifxe2x80x9d (J. Biol. Chem. (1926), 66, 375).
Proteins
*LOWRY (J. Biol. Chem. (1951), 193, 265-75).
Hexoses
*T. A. SCOTTxe2x80x94xe2x80x9cDosage colorimetr. a l""anthronexe2x80x9d (Anal. Chem. (1953), 25, 1956-61).
Hexosamines
*L. A. ELSON (Biochem. J. (1953), 27, 1824-28).
Lipopolysaccharides
*J. JANDA and E. WORK (Febs Letters, 1971, 16(4), 343-45).
Cxe2x80x94Other adiuvant factors of immunity, in addition to membranal fractions, are
of collagen type III
of sodium chloride
The Type III collagen used is characterized by:
axe2x80x94amino acid sequences similar to the following list (the concentrations are expressed in g/kg):
bxe2x80x94analysis type as follows:
The composition of the vaccinal complex according to the invention, associating RNA or ribosomal RNA fragments, membranal fractions (for example proteoglycanes of Klebsiella pneumoniae) and of type III collagen, completed by sodium chloride and an anti-inflammatory, permit, by administration of low doses giving rise to no toxicity, obtaining a high level of protection and of cure.
The preferred formulation is the injectable form of the composition described above, but it is possible to use other forms and/or other supports or additives compatible with medical usage.
The therapeutic vaccinal complex which has been described has the characteristics of a lymphokine which, adhering to the macrophages, inhibits the intra-cellular increase of the virus.
Since 1974-75 (A. S. and G. P. YOUMANS), it has been considered that the inhibitory effect of the immune response to RNA was produced by different inhibitors.
This has lead us to conceive the vaccinal complex, according to the invention, which gives rise to the same inhibition of the replication of the HIV retrovirus.
YOUMANS worked on a single bacterial strain (Mycobacterium tuberculosis), whose xe2x80x9cparasitismxe2x80x9d is solely intracellular.
VENNEMAN and colleagues believe, since 1972, that the true antigen could be associated with RNA, whose role would be that of an adjuvant. They vaccinated mice with ribosomal RNA, extracted with phenol at 65xc2x0 from ribosomes of the strain Salmonella typhimurium. Thirty days after this vaccination, they noted that the animals are better protected than by live vaccinal strain (attenuated).
It is above all believed that the level of protection is a function of the quantity of RNA injected.
It is now known that ribosomal RNA extract of Streptococcus pneumoniae produces a protection of humoral nature and that ribosomal RNA extract of Klebsiella pneumoniae induces a natural cellular protection.
Preliminary experiments, by DUSSOURD d""HINTERLAND, FONTANGES and colleagues (Division of Microbiology of the Research Center of the Military Health Service of Lyon, France), have thus demonstrated that this mixture, injected in vivo in mice and guinea pigs, has an action on the alveolar macrophages.
This xe2x80x9ctransitoryxe2x80x9d effect occurs when dosing the acid phosphatase, and the direct hemolyses regions when in contact with the splenic cells of mice.
Immunological studies carried out by DUSSORD d""HINTERLAND from ribosomal vaccines, induce in O. F. female mice the production of specific antibodies when they are administered to the animal in the presence of FREUND (incomplete) adjuvant or membranal protolycanes of Klebsiella.
The treatment with our vaccinal complex is as to itself followed by a cellular and humoral immunostimulant effect, with nonspecific action, but significant, on HIV. This action can be likened to the production of a xe2x80x9cV.N.F.xe2x80x9d (Virus Necrosis Factor), because it is the organism of the patient himself which is urged to reject the infected cells (antigen P24 being, for this reason, reduced to zero in almost all cases).
The therapeutic technique of S. ROSENBERG is not identical to ours, although it is rather close. He localized on human DNA the gene which produces the xe2x80x9cT.N.F.xe2x80x9d (Tumor Necrosis Factor). After having cloned on Escherichia coli and reproduced, he introduced it, with the aid of a vector virus, into lymphocytes T (Tueurs) which are found in tumors.
Our therapeutic mechanism permits producing a natural cloning thanks to the RNA (non-specific ribosomal bacteria) opsonized by the improved adjuvant (combination of membranal proteolycanes, of type III collagen, of sodium chloride).
This cloning induces a vaccination against idiotypes of the anti-HIV antibodies, as well as the production of antibodies against the site of bonding of the virus to the molecule CD4, in particular. To increase or inhibit the auto-immune anti-CD4 reaction (which contributes to lymphopeny CF4), it is necessary to use, during treatments with the vaccinal complex, corticoids (Betamethazone type) in the form of disodium phosphate, at a dosage of 20 to 60 mg, by the IV or IM route.
This action is also accompanied by a production of endogenous interferon, as well as an activation of the N. K. cells.
The object of our immunomodulatory vaccinal complex is therefore to induce an immune response having for its effect to prevent or at least to reduce (to a possible auto defense threshold), the proliferation of an infectious agent, viral in this case, introduced into the organism.
Our therapeutic novelty consists among other things in moderating or suppressing the existence of xe2x80x9csuppressive cellulesxe2x80x9d exerting a pro-infectious action.
Our treatment gives rise to an anti-AIDS reaction by a cellular and/or humoral defense response.
In conclusion, our therapeutic compound acts by directed evolution producing RNA molecules, which, binding to viral proteins, block the infection by AIDS retrovirus.
The vaccinal complex can be administered by oral route, but the preferred method is by the parenteral route:
either by direct intravenous direction
or by slow perfusion
or again by subcutaneous injection.
This various techniques have been carried out experimentally with success.
The daily dosages and their frequency depend greatly on the condition of the patient. An overdose has no risk, given the very low toxicity of the complex.
By intravenous route, can be used frequencies of one week per month, each day of the week of treatment comprising a slow perfusion of 500 ml of a solution containing:
0.9% sodium chloride
40 xcexcg of membranal saccharidic fractions (proteoglycanes of Klebsiella pneumoniae)
22 xcexcg (ribosomal) RNA of:
10 xcexcg of type III collagen described above
8 mg of disodium phosphate of Betamethazone (namely 2 ml of injectable solution)
This treatment by slow intravenous perfusion can be followed by a treatment by subcutaneous injections of the patients which can be carried out on an ambulatory basis, each injection containing
40 xcexcg of membranal saccharidic fractions (proteoglycanes of Klebsiella pneumoniae)
22 xcexcg (ribosomal) RNA of:
10 xcexcg of type III collagen described above
0.5 ml of 0.9% sodium chloride
4 mg of disodium phosphate of Betamethazone (namely 1 ml of injectable solution).
This treatment can be performed for several months, until the seropositivity is completely negated (that is to say AgP24 =0)
The following non-limiting examples are given to illustrate the concrete results of our therapeutic vaccinal complex.